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EP3600453A1 - Biomolécules radiomarquées et leur utilisation - Google Patents

Biomolécules radiomarquées et leur utilisation

Info

Publication number
EP3600453A1
EP3600453A1 EP18718637.4A EP18718637A EP3600453A1 EP 3600453 A1 EP3600453 A1 EP 3600453A1 EP 18718637 A EP18718637 A EP 18718637A EP 3600453 A1 EP3600453 A1 EP 3600453A1
Authority
EP
European Patent Office
Prior art keywords
compound
biomolecule
vhh
radiolabeled
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18718637.4A
Other languages
German (de)
English (en)
Inventor
Michael Rod Zalutsky
Ganesan Vaidyanathan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Duke University
Original Assignee
Duke University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Duke University filed Critical Duke University
Publication of EP3600453A1 publication Critical patent/EP3600453A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1051—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A61K51/1096—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/001—Acyclic or carbocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/004—Acyclic, carbocyclic or heterocyclic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen, sulfur, selenium or tellurium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic Table
    • C07F13/005—Compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention is drawn to compounds useful for radiolabeling biomolecules and to precursors thereof, as well as to radiolabeled biomolecules.
  • the compounds can effectively retain radioactivity from biomolecules that become internalized within cells, rendering such compounds useful in the diagnosis and treatment of disease, particularly cancer.
  • Radioiodination is one of the simplest ways to radiolabel a biomolecule.
  • Several radioisotopes of iodine are available for imaging and targeted radiotherapy of cancer. Radioisotopes of iodine are supplied as alkaline solutions and iodine is present in these in an oxidation state of -1 ( ⁇ ; iodide).
  • the standard method for biomolecule radioiodination requires oxidation of the iodine to the +1 oxidation state for electrophilic substitution into tyrosine amino acids present in biomolecules such as antibodies, other proteins and peptides.
  • radioiodinated monoclonal antibodies and peptides
  • mAbs monoclonal antibodies
  • peptides are proteolytically degraded inside cells after internalization (which can occur as a consequence of binding to receptors and certain antigens), to radioiodotyrosine that is efficiently exported from the cells by membrane amino acid transporters.
  • Radioiodotyrosine is deiodinated by deiodinases found in tissues and the free radioiodine redistributes and accumulates in organs with sodium iodide symporter expression, particularly the thyroid, stomach, and salivary glands. Thus, the amount of radiolabel that is retained in tumors is diminished and concomitantly, the uptake of radioactivity in normal tissues is increased.
  • the uptake of antibodies into tumor cells, particularly brain metastases, is low due to the size of the antibodies which is particularly problematic for tumors in the brain because of delivery restrictions imposed by the blood brain barrier.
  • the present invention addresses the problems associated with the treatment of cancer, including cancer that has metastasized to the brain by compositions that are capable of being taken up and retained by the tumor cells, while reducing the amount of the radiolabel that is taken up by normal tissue, particularly the kidneys.
  • the invention is drawn to methods, compounds, and compositions for radiolabeling biomolecules (also referred to as macromolecules) with radioactive halogen atoms in a manner which minimizes loss of the radioactive halogen due to dehalogenation in vivo, preserves the biological activity of the biomolecule, maximizes retention in diseased cells, such as cancer cells, and minimizes the retention of radioactivity in normal tissues after in vivo administration.
  • biomolecules have an affinity for particular types of cells. That is, the biomolecules may specifically bind a certain cell, such as cancer cells.
  • Compositions of the invention include the radiolabeled biomolecules.
  • biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles and aptamers.
  • biomolecules for purposes of the invention include, diabodies, scFv fragments, DARPins, fibronectin type Ill-based scaffolds, affibodies, VHH molecules (also, known as single domain antibody fragments (sdAb) and nanobodies), nucleic acid or protein aptamers, and nanoparticles.
  • larger molecules such as proteins >50 kDa including antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and F(ab') 2 fragments can be used in the practice of the invention.
  • nanoparticles with a size less than 50 nm can be used in the practice of the invention.
  • the methods of the invention utilize prosthetic compounds that are effective for radiolabeling.
  • the disclosure provides such radiolabeling compounds (referred to herein as “prosthetic compounds"), as well as precursors to afford such prosthetic compounds (referred to herein as “radiohalogen precursors").
  • the disclosure further provides radiolabeled macromolecules (e.g., biomolecules) comprising such prosthetic compounds/radicals and one or more macromolecules. In some such embodiments, these radiolabeled macromolecules are targeted radiotherapeutic agents.
  • the prosthetic compounds and radiolabeled compounds of the invention are useful, e.g., for diagnosing disease and for targeted radiotherapy.
  • X is CH or N
  • Lj and L 3 are independently selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain;
  • MMCM is a macromolecule conjugating moiety;
  • L 2 is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a polyethylene glycol (PEG) chain comprising at least three oxygen atoms, wherein L 2 optionally contains a Brush Border enzyme-cleavable peptide;
  • CG is selected from guanidine; P0 3 H; S0 3 H; one or more charged D- or L- amino acids, such as arginine, phosphono/sulfo phenylalanine, glutamate, aspartate, and lysine; a hydrophilic carbohydrate moiety; a polyethylene glycol (PEG) chain; and Z-guanidine (also referred to herein as "guanidino-Z");
  • Z is (CH 2 ) n ;
  • n is greater than 1 ;
  • Y is an alkyl metal moiety (in the radiohalogen precursor) or a radioactive halogen (in the prosthetic compound), wherein the radioactive halogen is selected from the group consisting of 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I 211 At, or a pharmaceutically acceptable salt or solvate thereof.
  • m 1.
  • Y is an alkyl metal moiety (where the compound is a radiohalogen precursor), selected from the group consisting of trimethyl stannyl (SnMe 3 ), tri-n-butylstannyl (SnBu 3 ) and trimethylsilyl (SiMe 3 ).
  • Y is a radioactive halogen (where the compound is a prosthetic compound) selected from the group consisting of 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I and 211 At.
  • MMCM is an active ester or (Gly) m , wherein m is 1 or more.
  • MMCM is selected from the group consisting of N-hydroxysuccinimide (NHS) ester, tetrafluorophenol (TFP) ester, an isothiocyanate group, or a maleimide group.
  • NHS N-hydroxysuccinimide
  • TFP tetrafluorophenol
  • isothiocyanate group or a maleimide group.
  • One exemplary MMCM is Gly-Gly-Gly.
  • the optional Brush Border enzyme-cleavable peptide, where present within L 2 is selected in some embodiments from the group consisting of Gly-Lys, Gly-Tyr and Gly-Phe-Lys.
  • the compound is represented by the following structure of Formula la:
  • the compound comprises N-succinimidyl 3-guanidinomethyl-5-
  • the disclosure provides a compound in the form of a prosthetic compound or radiohalogen precursor represented by Formula 2:
  • MC is a polydentate metal chelating moiety
  • Cm is thiourea, amide, or thioether
  • L 4 is selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, optionally having NH, CO, or S on one or both termini, and a polyethylene glycol (PEG) chain; and
  • T is a compound (prosthetic compound or radiohalogen precursor) as disclosed herein (e.g., according to Formula 1, e.g., Formula 1A),
  • MC is a macrocyclic structure.
  • MC is selected from DOTA, TETA, NOTP, and NOTA.
  • MC is an acyclic polydentate ligand.
  • MC is selected from EDTA, EDTMP, and DTPA.
  • Y is an alkyl metal moiety (where the compound is a radiohalogen precursor).
  • the alkyl metal moiety in the radiohalogen precursor is, for example, selected from the group consisting of trimethyl stannyl (SnMe 3 ), tri-n-butylstannyl (SnBu 3 ) and trimethylsilyl (SiMe 3 ).
  • SnMe 3 trimethyl stannyl
  • SnBu 3 tri-n-butylstannyl
  • SiMe 3 trimethylsilyl
  • Y is a radioactive halogen (where the compound is a prosthetic compound), such as 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I or 211 At.
  • the disclosure further provides a radiolabeled biomolecule, comprising a prosthetic compound as disclosed herein attached to a biomolecule and also provides an intermediate, comprising a radiohalogen precursor as disclosed herein attached to a biomolecule, which can be reacted to form a radiolabeled biomolecule.
  • the biomolecule can vary.
  • the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule, an aptamer or variations thereof.
  • the biomolecule is a VHH.
  • the VHH in particular embodiments, targets HER2.
  • the VHH comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1-5.
  • the disclosure further provides a pharmaceutical composition comprising a radiolabeled biomolecule as disclosed herein in association with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • a method of treatment for cancer comprising administering to an individual in need thereof an effective amount of a radiolabeled biomolecule as disclosed herein and/or an effective amount of a pharmaceutical composition as disclosed herein.
  • FIGURE 1 provides non-reducing SDS-PAGE/phosphor imaging profiles of (A) [ 211 At]SAGMB- 5F7 VHH, (B) [ 131 I]SGMIB-5F7 VHH, (C) «o-[ 211 At]SAGMB-5F7 VHH, and (D) «o-[ 131 I]SGMIB-5F7 VHH, with molecular weight standards in left lane for comparison;
  • FIGURE 2 provides the results of saturation binding assays performed on HER2 -expressing BT474M1 breast carcinoma cells with 5F7 VHH labeled using (A) [ 131 I]SGMIB, (B) wo-[ 131 I]SGMIB, (C) [ 211 At]SAGMB and (D) wo-[ 211 At] SAGMB;
  • FIGURE 3 provides plots of internalization of [ 211 At]SAGMIB-5F7 VHH and iso- [ 2U At] SAGMB - 5F7 VHH in BT474M1 cells in vitro, with FIG. 3 A depicting total cell-associated (internalized + surface- bound) radioactivity and FIG. 3B depicting internalized radioactivity;
  • FIGURE 4 provide plots of internalization of [ 131 I]SGMIB-5F7 VHH and wo-[ 131 I]SGMIB-5F7 VHH in BT474M1 cells in vitro, with FIG. 4A showing total cell-associated (internalized + surface-bound) radioactivity and FIG. 4B showing internalized radioactivity;
  • FIGURE 5 depicts biodistribution of [ 211 At]SAGMB-5F7 VHH and «o-[ 211 At]SAGMB-5F7 VHH in SCID mice bearing BT474M1 xenografts, with a comparison of uptake in tumor, with data obtained from paired-label studies after administering [ 131 I]SGMIB-5F7 / [ 2n At] SAGMB -5F7 VHH and wo-[ 131 I]SGMIB- 57 / «o-[ 211 At]SAGMB-5F7 VHH tandems;
  • FIGURE 6 depicts biodistribution of [ 131 I]SGMIB-5F7 VHH and «o-[ 131 I]SGMIB-5F7 VHH in SCID mice bearing BT474M1 xenografts: comparison of uptake in tumor, with data obtained from paired- label studies after administering [ 131 I]SGMIB-5F7 / [ 211 At]SAGMB-5F7 VHH and «o-[ 131 I]SGMIB-57 / iso- [ 211 At]SAGMB-5F7 VHH tandems;
  • FIGURE 7 depicts biodistribution of [ 211 At]SAGMB-5F7 and «o-[ 211 At]SAGMB-5F7 VHH in SCID mice bearing BT474M1 xenografts: comparison of uptake in kidneys, with data obtained from paired-label studies after administering [ 131 I]SGMIB-5F7 / [ 2n At] SAGMB -5F7 VHH and wo-[ 131 I]SGMIB- 57 / wo-[ 211 At]SAGMB-5F7 VHH tandems;
  • FIGURE 8 depicts biodistribution of [ 131 I]SGMIB-5F7 VHH and wo-[ 131 I]SGMIB-5F7 VHH in SCID mice bearing BT474M1 xenografts: comparison of uptake in kidneys, with data obtained from paired-label studies after administering [ 131 I]SGMIB-5F7 / [ 2n At] SAGMB -5F7 VHH and wo-[ 131 I]SGMIB- 57 / wo-[ 211 At]SAGMB-5F7 VHH tandems;
  • FIGURE 9 provides data on uptake of [ 2n At] SAGMB -5F7 VHH and wo-[ 211 At]SAGMB-5F7 VHH in thyroid (FIG. 9A) and stomach (FIG. 9B) in SCID mice bearing BT474M1 xenografts, with data obtained from paired-label studies after administering [ 131 I]SGMIB-5F7 / [ 2n At] SAGMB -5F7 VHH and iso- [ 131 I]SGMIB-57 / wo-[ 211 At]SAGMB-5F7 VHH tandems;
  • FIGURE 10 provides data on uptake of [ 131 I]SGMIB-5F7 and wo-[ 131 I]SGMIB-5F7 in thyroid (FIG.
  • FIGURE 11 depicts tumor-to-tissue ratios obtained from the biodistribution of [ 211 At]SAGMB-5F7 VHH and wo-[ 211 At]SAGMB-5F7 VHH in SCID mice bearing BT474M1 xenografts; with data obtained from paired-label studies after administering [ 131 I]SGMIB-5F7 / [ 211 At]SAGMB-5F7 VHH and iso- [ 131 I]SGMIB-5F7 / wo-[ 211 At]SAGMB-5F7 VHH tandems; and
  • FIGURE 12 depicts tumor-to-tissue ratios obtained from the biodistribution of [ 131 I]SGMIB-5F7 VHH and wo-[ 131 I]SGMIB-5F7 in SCID mice bearing BT474M1 xenografts, with data obtained from paired-label studies after administering [ 131 I]SGMIB-5F7/[ 211 At]SAGMB-5F7 VHH and wo-[ 131 I]SGMIB- 5F7 / wo-[ 211 At]SAGMB-5F7 VHH tandems;
  • FIGURE 13 is a table providing paired label biodistribution of [ 211 At]SAGMB-5F7 VHH and
  • FIGURE 14 is a table providing paired label biodistribution of wo-[ 211 At]SAGMB-5F7 VHH and ISO- [ 131 I]SGMIB-5F7 VHH in SCID mice with subcutaneous B474M1 human breast carcinoma xenografts.
  • compounds of the present disclosure comprise a radiolabeled prosthetic
  • radiolabeled prosthetic group attached to a macromolecule, e.g., a biomolecule that serves as a targeting moiety (providing a targeted radiotherapeutic agent).
  • a macromolecule e.g., a biomolecule that serves as a targeting moiety (providing a targeted radiotherapeutic agent).
  • the present disclosure encompasses radiolabeled prosthetic compounds and radicals themselves, as well as macromolecules having such radiolabeled prosthetic compounds/radicals attached thereto (which are referred to in some
  • the disclosure also encompasses such compounds and radicals (alone and/or in combination with a biomolecule) containing an alkyl metal moiety (referred to herein as "radiohalogen precursors") from which a prosthetic group and/or a targeted radiotherapeutic agent can be produced.
  • radiohalogen precursors an alkyl metal moiety
  • preparation of such precursors allows for the production of prosthetic compounds, as well as radioactive halogens (e.g., larger than 18 F, including, but
  • a labeled prosthetic compound/radical or a radiohalogen precursor (alone or attached to a macromolecule) generally includes, in addition to a radioactive halogen or precursor thereto, a charged group (CG), and a macromolecule conjugating moiety (MMCM).
  • CG charged group
  • MMCM macromolecule conjugating moiety
  • Each of these components can be associated with one or more cleavable (or non-cleavable) linkers, as will be described in more detail below.
  • the targeted radiotherapeutic agent in some embodiments, comprises a biomolecule (targeting moiety), a radiolabeled prosthetic group or template, and, optionally, a chelating agent (either macrocyclic or acyclic).
  • the radiolabeled compounds and, in particular, the radiolabeled biomolecules and the methods of use described herein, result in greater uptake of the radioactivity in the targeted cells, higher retention of radioactivity in the targeted cells after internalization, and less uptake of the radioactivity in normal cells; for example, there is less thyroid and renal uptake of the radioactivity.
  • the targeted radiotherapy of the invention is capable of selectively delivering a radionuclide to malignant cell populations.
  • An advantage of targeted radiotherapy is that one can select a radionuclide with properties that are best matched to the constraints of the intended clinical application.
  • CNS central nervous system
  • radiation would advantageously be selected with a tissue range that minimizes irradiation of normal CNS tissues.
  • the compounds provided herein are prepared by a method that enhances the retention of a radionuclide, particularly (in certain embodiments), a radiohalogen, in targeted diseased cells, such as cancer cells, using labeling techniques that generate a charged catabolite, following intracellular proteolysis, which cannot traverse the lysosomal or cell membrane and is resistant to exocytosis.
  • the compounds of the invention comprise a charged catabolite where the portion of the molecule bearing the label is inert to lysosomal degradation and becomes trapped inside the cell after proteolysis.
  • Certain prosthetic compounds and precursors thereto i.e., radiohalogen precursors encompassed by the present disclosure include those of Formula 1 and derivatives and variants thereof.
  • MMCM macromolecule conjugating moiety
  • Y radioactive halogen or a radiohalogen precursor
  • CG one or more charged substituents/groups
  • Each of these components can be attached to the aromatic ring through a linker (L l5 L 2 , L 3 ) or can be directly bonded to the aromatic ring (i.e., where Lj and/or L 2 and/or L 3 is a bond).
  • Lj and/or L 2 and/or L 3 is a bond
  • Y is a radioactive halogen (where Formula 1 represents a radiolabeled
  • radioactive halogens can be selected from 10 F, "Br, ,u Br, "Br, '”I, ⁇ %
  • the radioactive halogens in some embodiments are larger than F.
  • the radioactive halogen Y is selected from 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I, and 211 At.
  • the radioactive halogen Y is selected from 75 Br, 76 Br, 77 Br and 211 At.
  • the radioactive halogen Y is 211 At.
  • Y is an alkyl metal moiety (where Formula 1 represents a radiohalogen precursor/radical).
  • exemplary alkyl metal moieties include, but are not limited to, trialkyl metal precursors including trimethyl stannyl (SnMe 3 ), tri-n-butylstannyl (SnBu 3 ), and trimethylsilyl (SiMe 3 ).
  • L 3 can be, e.g., a spacer such as a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a short polyethylene glycol (PEG) chain (1 -10 ethylene glycol units).
  • the charged group is typically a group that is charged under the physiological conditions of the internal cell environment.
  • the charged group (CG) comprises a guanidine, a P0 H group, or an S0 H group.
  • CG is a guanidino-alkyl group containing more than one carbon.
  • CG is a guanidino-hydrophilic group (such as an amino- or hydroxyl- containing group), and/or an alkyloxycarbonylguanidine group.
  • CG comprises one or more charged D-amino acids such as arginine, glutamate, aspartate, lysine, and/or phosphono/sulfo phenylalanine.
  • CG comprises a hydrophilic carbohydrate moiety.
  • the compounds in some embodiments, may contain one, two or three CG moieties (and, optionally, corresponding linker groups L 2 ) to increase intracellular trapping in cancer cells.
  • L 2 can be, e.g., a spacer such as a substituted or unsubstituted alkyl chain (e.g., a simple substituted or unsubstituted alkyl chain such as a methylene), a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, a PEG chain of at least three oxygens, or any of the foregoing containing a Brush Border enzyme-cleavable peptide such as Gly-Lys, Gly-Tyr or Gly-Phe-Lys.
  • the unsubstituted alkyl chain comprises two or more carbon atoms.
  • a metabolizable spacer or cleavable linker L 2 (e.g., a Brush Border enzyme cleavable linker), is located between CG and the aromatic ring.
  • L 2 e.g., a Brush Border enzyme cleavable linker
  • linkers include linker sequences targeting meprin ⁇ , a metalloprotease expressed in the kidney brush-border membrane (Jodal et al. (2015) PLoS One Apr 9;10(4):e0123443); C-terminal lysines linked to antibody fragments via the epsilon-amino group of lysine or a C-terminal (N(epsilon)-amino-l,6-hexane-bis- vinyl sulfone)lysine that show reduced kidney uptake by taking advantage of the lysine specific
  • MMCM is an active ester.
  • An active ester is defined herein as an ester that can be conjugated with amine groups present on a macromolecule/biomolecule (e.g., a peptide or protein) under mild conditions, i.e., conditions that will not result in loss of biological function of the
  • MMCM groups include, but are not limited to, N- hydroxysuccinimide (NHS) or tetrafluorophenol (TFP) ester, an isothiocyanate group, or a maleimide group.
  • NHS N- hydroxysuccinimide
  • TFP tetrafluorophenol
  • Such MMCMs generally result in random (non-site specific) labeling of amine groups on the protein or peptide.
  • MMCM provides for site-specific conjugation to be performed using the enzyme Sortase, which results in conjugation to only one site (either the N-terminus or the C-terminus of the protein).
  • MMCM is, e.g., the tripeptide GlyGlyGly.
  • Lj can be, e.g., a spacer such as a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a short polyethylene glycol (PEG) chain (1-10 ethylene glycol units).
  • positions of these three moieties (-Lj-MMCM, -L 2 -CG, and -L 3 -Y) on the aromatic ring can vary. Where X is CH, these three moieties, can be placed at any of the positions of the aromatic ring. In some such embodiments, the -L 2 -CG, and -L 3 -Y moieties are located at the 3 and 4 positions, respectively (or the 4 and 3 positions, respectively) relative to the -Lj-MMCM moiety (at the 1 position).
  • the -L 2 -CG, and -L 3 -Y moieties are located at the 3 and 5 positions with respect to the -L MMCM moiety, such that the aromatic ring comprises the referenced moieties at the 1, 3, and 5 positions.
  • these three moieties can be placed at any of the remaining five positions of the ring, e.g., including, but not limited to, at the 2, 4, and 6 positions of the ring.
  • Certain prosthetic compounds within the scope of Formula 1 for labeling the targeting molecules of the invention, and radiohalogen precursors include compounds of Formula 1 A and derivatives and variants thereof, as shown below.
  • X is CH (i.e., the aromatic ring is a benzene ring)
  • L 2 is a methylene group
  • the three moieties (-Lj-MMCM, -L 3 -Y, and -CH 2 -CG) are present at the 1, 3, and 5 positions of the aromatic ring.
  • the invention also includes compounds thereof with the general structure of Formula 2 shown below (referred to as "Class II Type Compounds”).
  • Such compounds include a polydentate metal chelating moiety (MC), a linker (L 4 ) with a conjugating moiety (Cm) at both ends of L 4 , and a radiohalogenated template or radiohalogen precursor template (T).
  • T can be, for example, a compound of Formula 1 or a compound of Formula 1A, as shown above (a compound containing a MMCM).
  • T is a prosthetic compound/radical and in some embodiments, T is a radiohalogen precursor compound/radical.
  • m 0, where the "MC-Cm-L 4 -Cm" moiety of Formula 2 provides the desired function of the L 2 -CG moiety in Formula 1, above ⁇ i.e., the MC-Cm-L 4 -Cm substituent is a sufficiently "charged group”).
  • m 1, 2, or 3, such that the aromatic ring of "T” has at least four substituents, i.e., L MMCM, L 3 -Y, L 2 -CG, and Cm-L 4 -Cm-MC, and may optionally comprise one or more additional L 2 -CG substituents.
  • L 4 can be as defined above for Lj and L 3 .
  • L 4 can be a direct bond or can be, e.g., a spacer such as a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a short polyethylene glycol (PEG) chain (1-10 ethylene glycol units).
  • L 4 is again, as defined above but has NH, CO (carbonyl), or S (thioether) on one or both termini.
  • Cm can be, e.g., a thiourea, an amide, or a thioether.
  • Cm is thiourea (e.g., when the conjugating functionality in the chelating moiety and T is an isothiocyanate), an amide (when the conjugating functionality in the chelating moiety and T is NHS or TFP active ester, or acyl halide), or thioether (when the conjugating functionality in the chelating moiety and T is maleimide).
  • T is generally a radiolabeled moiety or a radiohalogen precursor containing a MMCM via which a macromolecule can be coupled to the compound.
  • T can, in some embodiments, be a compound/radical of Formula 1 or a compound/radical of Formula 1 A.
  • other radiohalogen templates can be used, including, but not limited to, /SO-SGMIB, as disclosed in Choi et al. (2014) Nucl Med Biol 41(10): 802-812, which is incorporated herein by reference; SIPC, as disclosed in Reist et al. (1997) Nucl Med Biol 24(7): 639-648, which is incorporated herein by reference; or SDMB, as disclosed in US Patent No. 5,302,700, which is incorporated herein by reference.
  • MC can be any polydentate moiety and can be cyclic or acyclic.
  • the composition of MC can vary.
  • MC can be either uncomplexed (lacking a metal) or complexed with the stable (nonradioactive) or radioactive form of a metal, preferably a trivalent metal (M +3 ) such as lutetium, yttrium, indium, actinium, or gallium and the MC is connected to the linker either using one of the free COOH groups present on the MC or via other positions on the MC including one of the MC backbone carbons.
  • M +3 trivalent metal
  • radioactive metals that can be complexed with the MC include, but are not limited to, radioactive metals selected from the group consisting of 177 Lu, 64 Cu, m In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr, and 227 Th. It is noted that this list is not exhaustive and, although these exemplified radioactive metals are trivalent, certain MCs that may be used according to the present invention may bind metals of other valencies, and such MCs and radioactive metals are also encompassed herein.
  • "T" may or may not include a radioactive atom (e.g., halogen).
  • T comprises a moiety as shown in Formula 1/la above, wherein the ' ⁇ " group is a non-radioactive halogen (e.g., a non-radioactive bromine or iodine).
  • a radiometal associated with MC, such as the radioactive metals referenced above.
  • such a strategy would allow, e.g., for use of the same prosthetic agent for multiple isotopes.
  • a compound of Formula 2 is provided with a low energy beta emitter (e.g., 131 I) plus a high energy beta emitter (e.g., 90 Y); or an alpha emitter (e.g.,
  • beta emitter halogen e.g., 131 I
  • alpha emitter halogen e.g., 211 At
  • beta emitter radiometal e.g., 177 Lu
  • MC is a macrocyclic ligand, consisting of a ring containing 8 or more atoms, bearing at least 3 negatively charged substituents such as carboxyl or phosphonate groups.
  • exemplary macrocyclic ligands suitable as the MC group include 1,4,7, 10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane- 1,4,8,11-tetraacetic acid (TETA), and l,4,7-triazacyclononane-l,4,7-tri(methylene phosphonic acid) (NOTP).
  • MC is MeO-DOTA, as disclosed in Gali et al., Anticancer Research (2001), 21(4A), 2785-2792), which is incorporated herein by reference.
  • Formula 2A Exemplary Class II Compound with DOTA MC
  • the left-hand brackets in Formula 2A are intended to convey that the specific site on the MC (DOTA) to which the Cm group is bonded is not limited, i.e. , the Cm may be bonded to DOTA at various sites thereon.
  • the right-hand brackets in Formula 2A are intended to convey that the specific site on the ring of "T" to which the Cm group is bonded is not limited, i.e., Cm may be bonded to T at various sites on the ring.
  • CG-L 2 may or may not be present.
  • the benzene ring of T in Formula 2A comprises four substituents (including the linked MC, L 2 -MMCM, L 3 -Y, and L 2 -CG). In other embodiments, the benzene ring of T in Formula 2 A comprises three substituents (including the linked MC, L 2 -MMCM, and L 3 -Y). The latter embodiments are particularly relevant when the linked MC is charged, i.e., it can take the place in providing the desired function of the "L 2 -CG" substituent.
  • MC is an acyclic ligand, consisting of a chain containing 6 or more atoms bearing at least 3 negatively charged substituents such as carboxyl or phosphonate groups.
  • exemplary acyclic ligands suitable as the MC group include diethylenetriaminepentaacetic acid (DTP A),
  • ethylenediaminetetramethylenephosphonic acid (EDTMP), and ethylenediaminetetraacetic acid (EDTA).
  • ETMP ethylenediaminetetramethylenephosphonic acid
  • EDTA ethylenediaminetetraacetic acid
  • An example of a Class II compound is illustrated below in Formula 2B, wherein MC is an acyclic ligand comprising DTPA, and wherein the radiohalogenated template T is a moiety corresponding to Formula 1.
  • Formula 2B Exemplary Class II Compound with DTPA (acyclic) MC
  • the left-hand brackets in Formula 2B are intended to convey that the specific site on the MC (DTPA) to which the Cm group is bonded is not limited, i.e. , the Cm may be bonded to DTPA at various sites thereon.
  • the right-hand brackets in Formula 2B are intended to convey that the specific site on the ring of "T" to which the Cm group is bonded is not limited, i.e. , Cm may be bonded to T at various sites on the ring.
  • CG-L 2 may or may not be present.
  • the benzene ring of T in Formula 2B comprises four substituents (including the linked MC, L 2 -MMCM, L 3 -Y, and L 2 -CG).
  • the benzene ring of T in Formula 2A comprises three substituents (including the linked MC, L 2 -MMCM, and L 3 -Y). The latter embodiments are particularly relevant when the linked MC is charged, i.e. , it can take the place in providing the desired function of the "L 2 -CG" substituent.
  • formulas above comprising a MMCM
  • an attached macromolecule e.g., biomolecule
  • compounds of any of the formulas provided herein above are encompassed, which further comprise a macromolecule (e.g., biomolecule) coordinated thereto via the MMCM.
  • the disclosure thus encompasses intermediates
  • radiolabeled biomolecules comprising a prosthetic group and a biomolecule, both of which may or may not comprise a metal chelating moiety.
  • the present disclosure further provides methods of synthesizing the prosthetic compounds and radiolabeled biomolecules described herein.
  • Employing such precursors allows for the preparation of prosthetic compounds and radiolabeled biomolecules comprising larger radioactive "Y" groups, e.g., larger than 18 F, including, but not limited to, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 At.
  • the macromolecule can be coordinated to the MMCM while Y is in the form of an alkyl metal radiohalogen precursor; then a subsequent reaction provides the product, wherein Y is in the form of the desired radioactive halogen atom.
  • C m -C n alkyl on its own or in composite expressions such as C m -C n haloalkyl, C m -C n alkylcarbonyl, C m -C n alkylamine, etc. represents a straight or branched aliphatic hydrocarbon radical having the number of carbon atoms designated, e.g. d-C 4 alkyl means an alkyl radical having from 1 to 4 carbon atoms.
  • Q- C 6 alkyl has a corresponding meaning, including also all straight and branched chain isomers of pentyl and hexyl.
  • Preferred alkyl radicals for use in the present invention are Q-Cealkyl, including methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, teri-butyl, n-pentyl and n-hexyl, especially Ci-C alkyl such as methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl and isobutyl. Methyl and isopropyl are typically preferred.
  • C 2 -C n alkenyl represents a straight or branched aliphatic hydrocarbon radical containing at least one carbon-carbon double bond and having the number of carbon atoms designated, e.g. C 2 -C 4 alkenyl means an alkenyl radical having from 2 to 4 carbon atoms; C 2 -C 6 alkenyl means an alkenyl radical having from 2 to 6 carbon atoms.
  • Non-limiting alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n- pentenyl and hexenyl.
  • C 2 -C n alkynyl represents a straight or branched aliphatic hydrocarbon radical containing at least one carbon-carbon triple bond and having the number of carbon atoms designated, e.g. C 2 -C 4 alkynyl means an alkynyl radical having from 2 to 4 carbon atoms; C 2 -C 6 alkynyl means an alkynyl radical having from 2 to 6 carbon atoms.
  • Non-limiting alkenyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl pentynyl and hexynyl.
  • An alkynyl group may be unsubstituted or substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, - NH 2 , -NH(alkyl), -N(alkyl) 2 , -NH(cycloalkyl), -0-C(0)-alkyl, -0-C(0)-aryl, -0-C(0)-cycloalkyl, -C(0)OH and -C(0)0-alkyl.
  • C m -C n haloalkyl represents C m -C n alkyl wherein at least one C atom is substituted with a halogen (e.g. the C m -C n haloalkyl group may contain one to three halogen atoms), preferably iodine, bromine, or fluorine.
  • Typical haloalkyl groups are C 1 _C 2 haloalkyl, in which halo suitably represents iodo.
  • Exemplary haloalkyl groups include iodomethyl, diiodomethyl and triiodomethyl. As used herein, only one of the halogens can be radioactive.
  • C m -C n hydroxyalkyl represents C m -C n alkyl wherein at least one C atom is substituted with one hydroxy group.
  • Typical C m -C n hydroxyalkyl groups are C m -C n alkyl wherein one C atom is substituted with one hydroxy group.
  • Exemplary hydroxyalkyl groups include hydroxymethyl and hydroxyethyl.
  • C m -C n alkylene represents a straight or branched bivalent alkyl radical having the number of carbon atoms indicated.
  • Preferred C m -C n alkylene radicals for use in the present invention are Q-Csalkylene.
  • alkylene groups include -CH 2 -, -CH 2 CH 2 -, - CH 2 CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )- and -CH(CH(CH 3 ) 2 )-.
  • Ci-C 4 alkoxy represents a radical C m -C n alkyl-0- wherein C m -C n alkyl is as defined above.
  • Ci-C 4 alkoxy which includes methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n- butoxy, sec-butoxy and isobutoxy. Methoxy and isopropoxy are typically preferred.
  • Ci-Cealkoxy has a corresponding meaning, expanded to include all straight and branched chain isomers of pentoxy and hexoxy.
  • Me means methyl
  • MeO means methoxy
  • amino represents the radical -NH 2
  • halo represents a halogen radical such as fluoro, chloro, bromo, iodo, or astato. Typically, halo groups are iodo, bromo or astato.
  • aryl represents an aromatic ring, for example a phenyl, biphenyl or naphthyl group.
  • heterocycloalkyl represents a stable saturated monocyclic 3-12 membered ring containing 1-4 heteroatoms independently selected from O, S and N. In one embodiment the stable saturated monocyclic 3-12 membered ring contains 4 N heteroatoms. In a second embodiment the stable saturated monocyclic 3-12 membered ring contains 2 heteroatoms independently selected from O, S and N. In a third embodiment the stable saturated monocyclic 3-12 membered ring contains 3 heteroatoms independently selected from O, S and N.
  • a heterocycloalkyl group may be unsubstituted or substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, - alkylene-O-alkyl, alkylthio, -NH 2 , -NH(alkyl), -N(alkyl) 2 , -NH(cycloalkyl), -0-C(0)-alkyl, -0-C(0)-aryl, - 0-C(0)-cycloalkyl, -C(0)OH and -C(0)0-alkyl. It is generally preferred that the heterocycloalkyl group is unsubstituted, unless otherwise indicated.
  • heteroaryl represents a stable aromatic ring containing 1 -4 heteroatoms independently selected from O, S and N.
  • heteroaryl moieties useful in the present disclosure have 6 ring atoms.
  • the stable aromatic ring system contains one heteroatom that is N.
  • aminoC m -C n alkyl represents a C m -C n alkyl radical as defined above which is substituted with an amino group, i.e. one hydrogen atom of the alkyl moiety is replaced by an NH 2 -group.
  • aminoC m -C n alkyl is aminoCj-Cealkyl.
  • aminoC m -C n alkylcarbonyl represents a C m -C n alkylcarbonyl radical as defined above, wherein one hydrogen atom of the alkyl moiety is replaced by an NH 2 -group.
  • aminoC m - C n alkylcarbonyl is aminoCj-Cealkylcarbonyl.
  • thio-monophosphate, thio-diphosphate and thio-triphosphate ester refers to groups:
  • radical positions on any molecular moiety used in the definitions may be anywhere on such a moiety as long as it is chemically stable. When any variable present occurs more than once in any moiety, each definition is independent.
  • solvates covers any pharmaceutically acceptable solvates that the compounds of Formula 1, and 2, as well as the salts thereof, are able to form.
  • Such solvates are, for example, hydrates, alcoholates, e.g., ethanolates, propanolates, and the like, especially hydrates.
  • Linkers may also be selected to facilitate bonding of the respective moieties to the core structure.
  • a representative linker is a Afunctional alkyl chain (e.g.,— CH 2 — ,— C 2 H 4 — ,— C 3 H 6 — , etc.) having from 1 to 6 carbon atoms, in which one carbon atom may be substituted with a cyclic (hydrocarbon ring) radical or heterocyclic (heterocyclic ring) radical.
  • Representative heterocyclic radicals have at least one nitrogen atom in the heterocyclic ring.
  • heterocyclic radicals are therefore diazinyl, diazolyl, triazinyl, triazolyl, tetrazinyl, and tetrazolyl radicals.
  • These and other heterocyclic radicals, or otherwise cyclic radicals may optionally be fused to a another cyclic or heterocyclic radical, or otherwise fused to a another cyclic or heterocyclic radical that is itself part of a fused ring system (e.g., a triazolyl radical may be fused to an 8-membered cyclic or heterocyclic radical that is itself fused to two 6-membered cyclic rings, as in the case of the triazolyl radical (or other nitrogen atom- substituted heterocyclic hydrocarbon radical) being fused to a dibenzoazocanyl radical).
  • a representative charged group linker, L 2 is a bivalent substituted or unsubstituted alkyl chain having from 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl chain, or a substituted or unsubstituted alkynyl chain.
  • L l 5 L 2 , L 3 and/or L 4 may be (or may comprise) substituted or unsubstituted bivalent alkyl radicals, having from 1 to 6 carbon atoms, wherein one or more carbon atoms may be substituted with and/or replaced by a heteroatom such as NH, O, or S, or otherwise may be substituted with or replaced by another alkyl radical (e.g., resulting in the formation of a branched alkyl radical) having from 1 to 8 carbon atoms that may be linear, branched, or cyclic.
  • a heteroatom such as NH, O, or S
  • a carbon-carbon double bond and/or a carbon-carbon triple bond may be formed between one or more pairs of adjacent carbon atoms, to provide bivalent, unsaturated (e.g., olefinic) alkyl radicals.
  • the targeted radiotherapy methods of the invention can utilize radiohalogens that emit radiations with ranges in tissue of less than 15 mm. These include alpha emitters such as 211 At, beta emitters such as 131 I and Auger electron emitters such as, 77 Br, 123 I, and 12 I, and the like.
  • Diagnostic imaging methods of the invention utilize radiations with ranges in tissue greater than 5 mm such that the radiation can be detected outside the body by positron emission tomography (PET) utilizing radiohalogens such as 75 Br, 76 Br, 124 I and the like; single photon emission computed tomography (SPECT) utilizing radiohalogens such as 123 I, 131 I, and 77 Br and the like; or intra-operative imaging that can be performed with any of the radiohalogens indicated above.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • intra-operative imaging that can be performed with any of the radiohalogens indicated above.
  • Theranostic methods of the invention utilize either 1) the same radiohalogen to perform targeted radiotherapy and diagnostic imaging (for example, 13 T, 123 1, 77 Br and the like) or 2) different radiohalogens of the same element to perform targeted radiotherapy and diagnostic imaging (for example, 124 I and 131 I; 123 I and 131 I; 77 Br and 76 Br; 77 Br and 75 Br; and the like).
  • other radiometals can be used, which bind to the metal chelate portion of the molecule.
  • biomolecules that may be coupled to radiolabeled prosthetic compounds described above include any molecule that specifically binds to a cell surface receptor, antigen or transporter.
  • Representative cell surface antigens or receptors include those that are internalized by the cell.
  • Biomolecules can be internalized by the cell over seconds, minutes, hours, or days. Preferred biomolecules are internalized rapidly, i.e., most of the biomolecule is internalized after minutes to hours.
  • a biomolecule is considered to bind specifically when it binds with an affinity constant (K D ) of 10 6 M or less, preferably 10 s M _1 or less.
  • a biomolecule can be an antibody, a fragment of an antibody, or a synthetic peptide that binds specifically to a cell surface antigen, receptor or transporter.
  • Antibodies include monoclonal antibodies (mAbs) and antibody fragments include VHH molecules (also known as single -domain antibody fragments (sdAbs) or nanobodies).
  • the biomolecule is an internalizing antibody or antibody fragment. Any antibody that specifically binds to a cell surface antigen and is internalized by the cell is an internalizing antibody.
  • the antibody can be an immunoglobulin of any class, i.e., IgG, IgA, IgD, IgE, or IgM, and can be obtained by immunization of a mammal such as a mouse, rat, rabbit, goat, sheep, primate, human or other suitable species, including those of the Camelidae family.
  • the antibody can be polyclonal, i.e., obtained from the serum of an animal immunized with a cell surface antigen or fragment thereof.
  • the antibody can also be monoclonal, i.e., formed by immunization of a mammal using the cell membrane or surface ligand or antigen or a fragment thereof, fusion of lymph or spleen cells from the immunized mammal with a myeloma cell line, and isolation of specific hybridoma clone, as is known in the art.
  • the antibody can also be a recombinant antibody, e.g., a chimeric or interspecies antibody produced by recombinant DNA methods.
  • a preferred internalizing antibody is a humanized antibody comprising human immunoglobulin constant regions together with murine variable regions which possess specificity for binding to a cell surface antigen (see, e.g., Reist et al., 1997). If a fragment of an antibody is used, the fragment should be capable of specific binding to a cell surface antigen.
  • the fragment can comprise, for example, at least a portion of an immunoglobulin light chain variable region and at least a portion of an immunoglobulin heavy chain variable region.
  • a biomolecule can also be a synthetic polypeptide which specifically binds to a cell surface antigen.
  • the biomolecule can be a synthetic polypeptide comprising at least a portion of an immunoglobulin light chain variable region and at least a portion of an immunoglobulin heavy chain variable region, as described in U.S. Pat. No. 5,260,203 or as otherwise known in the art.
  • B- cell lymphoma Press et al., 1994; Hansen et al., 1996), T-cell leukemia (Geissler et al., 1991) and neuroblastoma cells (Novak-Hofer et al., 1994) all possess antigens that are internalized rapidly.
  • Internalizing receptors have been used to target mAbs to tumors. These include wild-type epidermal growth factor receptor (EGFR; gliomas and squamous cell carcinoma; Brady et al., 1992; Baselga et al., 1994), the pi 85 c-erbB-2 oncogene product, HER2 (breast and ovarian carcinomas; De Santes et al. 1992; Xu et al., 1997), and the transferrin receptor (gliomas and other tumors; Laske et al., 1997). Indeed, it has been suggested that internalization can occur with virtually any mAb that binds to a cell-surface antigen (Mattes et al., 1994; Sharkey et al., 1997a).
  • An advantage of mAb internalization for radioimmunotherapy is the potential for increasing the radiation absorbed dose delivered to the cell nucleus provided that the radioactivity is trapped on the targeted cell for a prolonged period.
  • Radiation dosimetry calculations suggest that even with the multicellular range ⁇ -emitter 13 T, shifting the site of decay from the cell membrane to cytoplasmic vesicles could increase the radiation dose received by the cell nucleus by a factor of two (Daghighian et al., 1996), thereby potentially increasing treatment.
  • a disadvantage of mAb internalization is that this event exposes the mAb to additional catabolic processes that can result in the release of radioactivity from the tumor cell, decrease the radiation dose to cancer cells and increasing the radiation dose to normal tissues in the body.
  • Antigens or receptors that are internalized by the cell can eventually become localized within endosomes or lysosomes.
  • the targeting moiety or internalization moiety are moieties that bind to the targeted diseased cells, such as cancer cells, and are internalized after binding to a cell surface receptor, a transporter, antigens found on the cell surface such as, for example, transmembrane receptors, extracellular growth factors, etc.
  • the compounds of the invention can be directed to any population of diseased cells or tumor cells.
  • it can be broadly used to target any cancer, tumor, or malignant growth.
  • the compounds of the invention can be targeted to human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), its tumor-specific mutant EGFRvIII , vascular endothelial growth factor (VEGF), VEGFA/B, EGFR (HER1/ERBB1), HER2 (ERBB2/neu), ALK, Axl, CD20, CD30, CD38, CD47, CD52, CDK4, CDK6, PD-1, PD-L1, KIT, VEGFR1/2/3, BAFF, HDAC, Proteasome, ABL, FLT3, KIT, MET, RET, IL-6, IL-6R, IL- ⁇ , EGFR(HER1/ERBB 1), MEK, ROS1, BRAF, ABL, RANKL, B4GALNT1(GD2), SLAMF7, (CS1/CD319/CRACC), mTOR, BTK, P13K5, PDGFR, PDGFRa, PDGFR , CTLA4, PARP, HDAC,
  • the targeting moiety can be selected from anti-HER2 VHH sequences such as those set forth in SEQ ID NOS: 1-5 and fragments and variants thereof that retain the binding specificity of the sequences. That is, the invention encompasses fragments, analogs, mutants, variants, and derivatives of the radiolabeled VHH domains. These oligoclonal VHHs are able to target a range of different epitopes on the HER2 receptor. Some of the VHHs do not compete with trastuzumab for binding on HER2.
  • the fragment, analog, mutant, variant and/or derivative of the VHH sequences provided herein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with at least one of SEQ ID NOS: 1-5. See Table 1.
  • amino acid sequences and nucleic acid sequences are exactly the same if they have 100% sequence identity over their entire length.
  • X% identical refers to absolute percent identity, unless otherwise indicated.
  • absolute percent identity refers to a percentage of sequence identity determined by scoring identical amino acids or nucleic acid as one and any substitution as zero, regardless of the similarity of mismatched amino acids or nucleic acids. In a typical sequence alignment, the "absolute percent identity" of two sequences is presented as a percentage of amino acid or nucleic acid “identities”.
  • Gaps can be internal or external, i.e., a truncation. Absolute percent identity can be readily determined using, for example, the Clustal W program, version 1.8, June 1999, using default parameters (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680).
  • the radiolabeled biomolecules of the invention can be targeted to any diseased or malignant cell population. In some instances, it may be preferred to use small biomolecules.
  • Brain metastases are cancer cells that have spread to the brain from primary tumors in other organs in the body. Metastatic tumors are among the most common mass lesions in the brain. An estimated 24-45% of all cancer patients have brain metastases. Lung, breast, melanoma, colon, and kidney cancers commonly spread to the brain. Brain metastases are associated with poor survival and high morbidity. Improving therapies for metastatic brain tumors is an aspect of the present invention.
  • the calculated pore size of a brain metastasis of breast cancer is less than 10 nm in diameter.
  • the targeting biomolecules of the invention are small molecules, including, but not limited to, affibodies, designed ankyrin repeat proteins (DARPins), aptamers, and VHH molecules (also known as single domain antibody fragments (sdAb) or nanobodies), collectively called small biomolecules herein.
  • DARPins ankyrin repeat proteins
  • VHH molecules also known as single domain antibody fragments (sdAb) or nanobodies
  • small biomolecules also known as single domain antibody fragments (sdAb) or nanobodies
  • Other "small molecule” scaffolds are characterized by mass/size, e.g., less than 10 nm in size or less than 25kDa. As indicated, these small biomolecules are designed to bind to a portion of the cancer cells.
  • VHHs can be prepared to specifically bind receptors on the cancer cells, such as human epidermal growth factor receptor-2 (HER2) or any of the other receptors listed above.
  • HER2 human epidermal growth factor receptor-2
  • VHHs Due to their small size, VHHs, aptamers and other small biomolecules diffuse and distribute efficiently throughout solid tumors, and due to their high binding specificity and affinity to their target antigens, high tumor uptake of the small biomolecules can be observed. Importantly, their half-life in the bloodstream is significantly shorter than full-length antibodies or larger targeting proteins, allowing rapid clearance of the unbound fraction of the small biomolecule by the kidneys, leading to higher tumor -to- normal tissue ratios shortly after their administration. VHHs are easily generated in nanomolar to picomolar affinity by cloning from immunized camels or llamas and selection by phage display panning.
  • VHHs or sdAb are stable and easily produced in large quantities using industrial grade methods and qualified bacteria, yeast, or mammalian cells. Compared with other small protein-based targeting vectors, VHHs generally offer significant advantages in terms of stability, solubility, expression yields, construction of multimers, as well as the ability to recognize hidden or uncommon epitopes. See, US Patent Nos:
  • Aptamers are oligonucleotide or peptide molecules that bind to a specific target molecule.
  • Aptamers can be nucleic acid molecules (DNA, RNA, XNA) and consist of short strands of oligonucleotides, peptide molecules that consist of one or more short variable peptide domains.
  • Aptamers offer molecular recognition properties readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. See, Keefe et al. (2010) Nature Reviews Drug Discovery 9:537-550; Ellington and Szostak (1990) Nature 346:818-822; Tuerk and Gold (1990) Science 249:505-510; Kulbachinskiy, A. V. (2007) Biochemistry 72: 1505-1518; all of which are herein incorporated by reference.
  • the '(calculated mean) effective dose' of radiation within a subject refers to the tissue-weighted sum of the equivalent doses in all specified tissues and organs of the body. It takes into account the type of radiation and the nature of each organ or tissue being irradiated. It is the central quantity for dose limitation in radiological protection in the international system of radiological protection devised by the International Commission on Radiological Protection (ICRP).
  • the SI unit for effective dose is the Sievert (Sv) which is one joule/kilogram (J/kg).
  • the effective dose replaced the former "effective dose equivalent" in 1991 in the ICRP system of dose quantities.
  • the effective dose is typically expressed per unit of injected activity, i.e. expressed in mSv/MBq.
  • the effective dose for the individual patient will then depend upon the injected activity of the radiopharmaceutical, expressed in MBq, and the calculated mean effective dose, expressed in mSv/MBq.
  • the effective dose for radiopharmaceuticals is calculated using OLINDA/EXM® software that was approved in 2004 by the FDA.
  • OLINDA/EXM® personal computer code performs dose calculations and kinetic modeling for radiopharmaceuticals (OLINDA/EXM stands for Organ Level Internal Dose Assessment/Exponential Modeling).
  • OLINDA® calculates radiation doses to different organs of the body from systemically administered radiopharmaceuticals and performs regression analysis on user-supplied biokinetic data to support such calculations for nuclear medicine drugs. These calculations are used to perform risk/benefit evaluations of the use of such pharmaceuticals in diagnostic and therapeutic applications in nuclear medicine.
  • the technology employs several standard body models for adults, children, pregnant women and others, that are widely accepted and used in the internal dose community. The calculations are useful to pharmaceutical industry developers, nuclear medicine professionals, educators, regulators, researchers and others who study the accepted radiation doses that should be delivered when radioactive drugs are given to patients or research subjects.
  • the calculated effective dose depends on the chosen standard body model and the chosen voiding bladder model.
  • the values provided herein have been calculated using the female adult model and a voiding bladder interval of 1 h.
  • the prevention and/or treatment of cancer is achieved by
  • a radiolabeled small biomolecule i.e., an aptamer, VHH or functional fragments thereof, and the like, as disclosed herein to a subject in need thereof, characterized in that the small biomolecule has a calculated mean effective dose of between 0.001 and 0.05 mSv/MBq in a subject, such as but not limited to a calculated mean effective dose of between 0.02 and 0.05 mSv/MBq, more preferably between 0.02 and 0.04 mSv/MBq, most preferably between 0.03 and 0.05 mSv/MBq.
  • the dose of radioactivity applied to the patient per administration must be high enough to be effective but must be below that which would result in dose limiting toxicity (DLT).
  • DLT dose limiting toxicity
  • compositions comprising radiolabeled antibodies, e.g. with 131 Iodine, the maximally tolerated dose (MTD) must be determined which must not be exceeded in therapeutic settings.
  • MTD maximally tolerated dose
  • biomolecules The proteins and peptides (collectively referred to as biomolecules below) as envisaged herein and/or the compositions comprising the same are administered according to a regimen of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated.
  • the clinician will generally be able to determine a suitable treatment regimen.
  • the treatment regimen will comprise the administration of one or more small biomolecules, such as VHH sequences or polypeptides, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses.
  • the desired dose may conveniently be presented in a single dose or as divided doses (which can again be sub-dosed) administered at appropriate intervals.
  • An administration regimen could include long- term (i.e., at least two weeks, and for example several months or years) or daily treatment.
  • an administration regimen can vary between once a day to once a year, such as between once a day and once every twelve months, such as but not limited to once a week.
  • pharmaceutical small biomolecule compositions as disclosed herein may be administered once or several times, also intermittently, for instance daily for several days, weeks or months and in different dosages.
  • the amount applied of the small biomolecule compositions disclosed herein depends on the nature of the cancer or other disease to be treated.
  • radiolabeled materials are typically administered at intervals of 4 to 24 weeks apart, preferably 8 to 20 weeks apart. The skilled artisan knows how to divide the administration into two or more applications, which may be applied shortly after each other, or at some other predetermined interval ranging e.g. from 1 day to 1 week.
  • biomolecules disclosed herein may be used in combination with other biomolecules disclosed herein.
  • both treatments are applied to the patient in temporal proximity.
  • both treatments are applied to the patient within four weeks (28 days). More preferably, both treatments are applied within two weeks (14 days), more preferred within one week (7 days).
  • the two treatments are applied within two or three days.
  • the two treatments are applied at the same day, i.e. within 24 hours.
  • the two treatments are applied within four hours, or two hours, or within one hour.
  • the two treatments are applied in parallel, i.e. at the same time, or the two administrations are overlapping in time.
  • the radiolabeled biomolecules of the invention are applied together with one or more therapeutic antibodies or therapeutic antibody fragments.
  • the targeted radiotherapy with the radiolabeled biomolecule is combined with regular immunotherapy with one or more therapeutic antibodies or therapeutic antibody fragments.
  • the radiolabeled biomolecules are used in a combination therapy or a combination treatment method with one or more therapeutic antibodies or therapeutic antibody fragments, such as but not limited to a combination treatment with Trastuzumab (Herceptin®) and/or Pertuzumab (Perjeta®).
  • the radiolabeled biomolecules and the one or more therapeutic antibodies or therapeutic antibody fragments may be infused at the same time, or the infusions may be overlapping in time.
  • the two drugs may be formulated together in one single pharmaceutical preparation, or they may be mixed together immediately before administration from two different pharmaceutical preparations, for example by dissolving or diluting into one single infusion solution.
  • the two drugs are administered separately, i.e. , as two independent pharmaceutical compositions.
  • administration of the two treatments is in a way that tumor cells within the body of the patient are exposed to effective amounts of the cytotoxic drug and the radiation at the same time.
  • effective amounts of both the radiolabeled biomolecules of the invention and the one or more therapeutic antibodies or therapeutic antibody fragments such as but not limited to Trastuzumab (Herceptin®) and/or Pertuzumab (Perjeta®) are present at the site of the tumor at the same time.
  • the present invention also embraces the use of further agents, which are administered in addition to the combination as defined. This could be, for example, one or more further chemotherapeutic agent(s).
  • a cytokine stimulating proliferation of leukocytes may be applied to ameliorate the effects of leukopenia or neutropenia.
  • the use of the radiolabeled biomolecules as envisaged herein that specifically bind to a tumor-specific or cancer cell-specific target molecule of interest is provided for the preparation of a medicament for the prevention and/or treatment of at least one cancer -related disease and/or disorder in which said tumor-specific or cancer cell-specific target molecule is involved.
  • the application provides biomolecules specifically binding to a tumor-specific or cancer cell-specific target, such as those set forth above, for use in the prevention and/or treatment of at least one cancer -related disease and/or disorder in which said tumor-specific or cancer cell-specific target is involved.
  • methods for the prevention and/or treatment of at least one cancer-related disease and/or disorder comprising administering to a subject in need thereof, a pharmaceutically active amount of one or more biomolecules including VHH sequences or functional fragments thereof, polypeptides, aptamers, etc., and/or pharmaceutical compositions as envisaged herein.
  • the subject or patient to be treated with the radiolabeled biomolecules described herein may be any warm-blooded animal, but is in particular, a mammal and more particularly, a human suffering from, or at risk of, a cancer-related disease and/or other disease disorder.
  • the efficacy of the biomolecules, i.e., VHH sequences or functional fragments thereof, aptamers, polypeptides, and the like described herein, and of compositions comprising the same can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person.
  • the skilled person will generally be able to select a suitable in vitro assay, cellular assay or animal model to test the biomolecules described herein for binding to the tumor-specific or cancer cell-specific molecule; as well as for their therapeutic and/or prophylactic effect in respect of one or more cancer-related diseases and disorders.
  • biomolecules comprising or essentially consisting of at least one radiolabeled biomolecule or functional fragments thereof for use as a medicament, and more particularly for use in a method for the treatment of a disease or disorder related cancer, including solid tumors.
  • the radiolabeled biomolecules envisaged herein are used to treat and/or prevent cancers and neoplastic conditions.
  • cancers or neoplastic conditions include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma
  • proliferative disorders include hematopoietic neoplastic disorders and cellular proliferative and/or differentiative disorders, such as but not limited to, epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, miscellaneous
  • Imaging of radioactivity after administration of the biomolecule labeled with the claimed prosthetic compounds can be performed by standard radiological methods, including, for example, scanning the body with a gamma camera (radioscintigraphy), single photon emission computed tomography (SPECT) and positron emission tomography (PET) (see, e.g., Bradwell et al., Immunology Today 6: 163-170, 1985).
  • SPECT single photon emission computed tomography
  • PET positron emission tomography
  • the labeled prosthetic compound, coupled to a biomolecule should be given in either diagnostically or therapeutically acceptable amounts.
  • a therapeutically acceptable amount is an amount which, when given in one or more dosages, produces the desired therapeutic effect, e.g., shrinkage of a tumor, with a level of toxicity acceptable for clinical treatment.
  • Such an administered amount will cause sufficient radiation to absorb within tumor cells so as to damage these cells, for example by disrupting their DNA.
  • Such an administered amount preferably should cause minimal damage to neighboring and distant healthy cells.
  • Both the dose of a particular composition and the means of administering the composition can be determined based on specific qualities of the composition, the condition, age, and weight of the patient, the progression of the particular disease being treated, and other relevant factors. If the composition contains antibodies, effective dosages of the composition are in the range of about 5 ⁇ g to about 50 ⁇ g/kg of patient body weight, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg.
  • a diagnostically acceptable amount of radioactivity is an amount which permits detection of radioactivity from the labeled biomolecule as required for diagnosis, with a level of toxicity acceptable for diagnosis.
  • Embodiment 1 A compound represented by Formula I (including prosthetic compounds and radiohalogen precursors):
  • X is CH or N
  • Lj and L 3 are independently selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain;
  • MMCM is a macromolecule conjugating moiety
  • L 2 is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a polyethylene glycol (PEG) chain comprising at least three oxygen atoms, wherein L 2 optionally contains a Brush Border enzyme-cleavable peptide;
  • CG is selected from guanidine, P0 3 H, S0 3 H, one or more charged D-amino acids, arginine or phosphono/sulfo phenylalanine, glutamate, aspartate, lysine, a hydrophilic carbohydrate moiety, a polyethylene glycol (PEG) chain, and guanidino-Z;
  • Z is (CH 2 ) n ;
  • n is greater than 1 ;
  • Y is an alkyl metal radiohalogen precursor or a radioactive halogen selected from the group consisting of 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I, and 211 At, or a pharmaceutically acceptable salt or solvate thereof.
  • Embodiment 2 The compound of Embodiment 1, wherein Y is an alkyl metal radiohalogen precursor selected from the group consisting of trimethyl stannyl (SnMe 3 ), tri-n-butylstannyl (SnBu 3 ) and trimethylsilyl (SiMe 3 ).
  • Y is an alkyl metal radiohalogen precursor selected from the group consisting of trimethyl stannyl (SnMe 3 ), tri-n-butylstannyl (SnBu 3 ) and trimethylsilyl (SiMe 3 ).
  • Y is a radioactive halogen selected from
  • Embodiment 4 The compound of any of Embodiment s 1-3, wherein MMCM is an active ester or (Gly) m , wherein m is 1 or more.
  • Embodiment 5 The compound of any one of Embodiments 1-3, wherein MMCM is selected from the group consisting of N-hydroxysuccinimide (NHS), tetrafluorophenol (TFP) ester, an isothiocyanate group, or a maleimide group.
  • NHS N-hydroxysuccinimide
  • TFP tetrafluorophenol
  • Embodiment 6 The compound of any one of Embodiments 1-3, wherein MMCM is Gly-Gly-Gly.
  • Embodiment 8 The compound of any one of Embodiments 1-7, wherein the optional Brush Border enzyme-cleavable peptide is selected from the group consisting of Gly-Lys, Gly-Tyr and Gly-Phe-Lys.
  • Embodiment 9 The compound of any of Embodiments 1-8, represented by the following structure:
  • Embodiment 10 The compound of Embodiment 9, wherein the compound is N-succinimidyl 3- guanidinomethyl-5-[ 131 I]iodobenzoate, or N-succinimidyl 3-[ 2n At]astato-5-guanidinomethyl benzoate.
  • Embodiment 11 A radiolabeled biomolecule or intermediate, comprising the compound of any one of Embodiments 1-10 attached to a biomolecule.
  • Embodiment 12 The radiolabeled biomolecule or intermediate of Embodiment 11, wherein the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule, an aptamer or variations thereof.
  • Embodiment 13 The radiolabeled biomolecule or intermediate of Embodiment 11 or 12, wherein said labeled biomolecule is a VHH.
  • Embodiment 14 The radiolabeled biomolecule or intermediate of Embodiment 13, wherein said VHH targets HER2.
  • Embodiment 15 The radiolabeled biomolecule or intermediate of Embodiment 14, wherein said VHH comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1-5.
  • Embodiment 16 A pharmaceutical composition comprising the radiolabeled biomolecule of any of Embodiments 11-15 (where the compound is in the form of a prosthetic compound) in association with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • Embodiment 17 A compound represented by Formula 2 (including prosthetic compounds and radiohalogen precursors):
  • MC is a poly dentate metal chelating moiety
  • Cm is thiourea, amide, or thioether
  • L 4 is selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, optionally having NH, CO, or S on one or both termini, and a polyethylene glycol (PEG) chain; and
  • T is the compound of any of Embodiments 1-10,
  • Embodiment 18 The compound of Embodiment 17, wherein MC is a macrocyclic structure.
  • Embodiment 19 The compound of Embodiment 17, wherein MC is selected from DOTA, TETA, NOTP, and NOTA.
  • Embodiment 20 The compound of Embodiment 17, wherein MC is an acyclic polydentate ligand.
  • Embodiment 21 The compound of Embodiment 17, wherein MC is selected from EDTA, EDTMP, and DTPA.
  • Embodiment 22 The compound of any one of Embodiments 17-21, further comprising a metal associated with the MC.
  • Embodiment 23 The compound of Embodiment 21, wherein the metal is a radioactive metal selected from the group consisting of 177 Lu, 64 Cu, m In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr, and 227 Th
  • Embodiment 24 The compound of any one of Embodiments 17-23, wherein Y is an alkyl metal moiety (and the compound is a radiohalogen precursor).
  • Embodiment 25 The compound of Embodiment 24, wherein the alkyl metal moiety is selected from the group consisting of trimethyl stannyl (SnMe 3 ), tri-n-butylstannyl (SnBu 3 ) and trimethylsilyl (SiMe 3 ).
  • Embodiment 26 The compound of any one of Embodiments 17-23, wherein Y is a radioactive halogen, such as 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I, or 211 At (and the compound is a prosthetic compound).
  • Y is a radioactive halogen, such as 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I, or 211 At (and the compound is a prosthetic compound).
  • Embodiment 27 A radiolabeled biomolecule or intemediate, comprising the compound of any one of Embodiments 17-26, attached to a biomolecule.
  • Embodiment 28 The radiolabeled biomolecule or intermediate of Embodiment 27, wherein the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule and an aptamer.
  • Embodiment 29 The radiolabeled biomolecule or intermediate of Embodiment 27, wherein said labeled biomolecule is a VHH.
  • Embodiment 30 The radiolabeled biomolecule or intermediate of Embodiment 29, wherein said VHH targets HER2.
  • Embodiment 31 The radiolabeled biomolecule or intermediate of Embodiment 30, wherein said VHH comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1 -5.
  • Embodiment 32 A pharmaceutical composition comprising the radiolabeled biomolecule of any of Embodiments 27-31 (wherein the compound is a prosthetic compound), in association with a
  • Embodiment 33 A method of treatment for cancer comprising administering to an individual in need thereof an effective amount of the radiolabeled biomolecule of any one of Embodiments 11-15 or 27- 31 or an effective amount of the pharmaceutical composition of claim or Embodiment 16 or 32.
  • the disclosure includes any combination of two, three, four, or more of the above-noted embodiments as well as combinations of any two, three, four, or more features or elements set forth in this disclosure, regardless of whether such features or elements are expressly combined in a specific embodiment herein.
  • a molecule containing the guanidine -bearing amino acid arginine, Brush Border enzyme-cleavable linker dipeptide GlyTyr, and connected to the SIB moiety via a PEG linker (Arg-Gly-Tyr-PEG-SIB), is shown below in Schemes 4-6.
  • the radiolabeled version of this molecule for example, Arg-Gly-Tyr-PEG- [ 131 I]SIB, is obtained from the corresponding tin precursor using a standard iododestannylation reaction.
  • PEG diamine can be anchored to a trityl chloride resin and the three amino acids can be attached sequentially.
  • the resultant peptide derivative (1 mmol) is reacted with bis(2,5-dioxopyrrolidin-l-yl) 5-iodoisophthalate (486.2 mg; 1 mmol) in a mixture of THF and 0.1 M sodium carbonate buffer, pH 8.5.
  • the scheme for the synthesis of DOTA-PEG-SIB is shown in Scheme 7.
  • the same approach can be used to synthesize its tin precursor.
  • the tin precursor can be labeled with radioiodine using standard conditions; the DOTA moiety present in both the iodo and tin derivatives can be complexed with nonradioactive lutetium.
  • SIB-DOTA Vaidyanathan et al. (2012) Bioorg. Med. Chem. 20(24):6929-6939
  • all four COOH groups in the DOTA macrocycle are available to complex with a metal ion and the PEG linker replaces the hydrophobic 6-carbon alkyl chain.
  • the linker could include a Brush Border cleavable amino acid sequence. ' NHBOG
  • Trifluoroacetic acid 300 ⁇ is added to the above product (16 mg, 16 ⁇ ) and the resultant solution stirred at 20°C overnight. TFA is evaporated to give 2,2',2"-(10-(l-amino-16-carboxy-13-oxo-3,6,9-trioxa- 12-azahexadecan-16-yl)-l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid as an oil (lOmg, 15.4 ⁇ , 96% yield). LRMS (LCMS-ESI) m/z: 651.3 (M+H) + .
  • the above product is coupled to bis(2,5- dioxopyrrolidin-l-yl) 5-iodoisophthalate by reacting one equivalent of each reagent as well as one equivalent of N,N-diisopropylethylamine in DMF.
  • the product is purified by reversed-phase HPLC and conjugated with a macromolecule for subsequent labeling with a radiometal such as 177 Lu.
  • the previous examples illustrate approaches that consist of first synthesizing the radiohalogenated molecule (from a tin or other alkylmetal precursor) and then coupling the radiolabeled molecule to a macromolecule.
  • the alternative approach is to first react the precursor for radiohalogen with the macromolecule and then radiolabel this protein-precursor conjugate.
  • This second approach is called preconjugation and has several potential advantages including decreasing synthesis time (important with radioactivity) and increasing overall yields.
  • the tin-containing precursor molecule is first conjugated to the macromolecule. Then such derivatized macromolecules can be radiohalogenated, with this procedure preferably being performed at a pH lower than 6.5. This approach is illustrated i m).
  • the iodo derivative with an uncomplexed DOTA moiety can be used for labeling with radiometals such as 177 Lu.
  • radiometals such as 177 Lu.
  • 177 LuCl 3 2 Ci/ml, 10 ⁇ in 0.05 M HC1 is diluted with 0.15 M ammonium acetate buffer and reacted with 100-1000 ⁇ g of DOTA-PEG-SIB and when the reaction has run to completion, purified by standard size exclusion chromatography methods.
  • the tin derivative with the uncomplexed DOTA moiety can be conjugated with the macromolecule and then can be labeled with both a radiometal and a radiohalogen.
  • the DOTAGA derivative 2,2',2"-(10-(l-amino-16-carboxy-13-oxo-3,6,9-trioxa-12-azahexadecan- 16-yl)-l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid is coupled to bis(2,5-dioxopyrrolidin-l-yl) 5- (trimethylstannyl)isophthalate following the same procedure described above for the iodo derivative. It is then complexed with non-radioactive lutetium.
  • tin derivative 50 ⁇ of the tin derivative is treated with 5 equivalents of LuCl 3 in 10 ml of 0.4 M acetate buffer, pH 5.2.
  • the progress of the reaction is followed by reversed-phase HPLC and the lutetium complex is purified by semi-preparative reversed-phase HPLC.
  • the complex is then conjugated to a macromolecule.
  • a solution of the macromolecule in 0.2 M sodium carbonate buffer, pH 8.5 (10 nmol/ml) is added to a solution of the prosthetic agent in DMSO (25 mM; 5 ⁇ , 125 nmol), and the mixture incubated at 20°C for 1 h.
  • the resultant macromolecule -prosthetic group conjugate is isolated and at the same time buffer exchanged to 0.2 M acetate, pH 5.5, by filtering through a VivaSpin ultra filtration unit with appropriate molecular weight cut off (for example, 10 kDa for VHH) (GE Healthcare).
  • the modified macromolecule is then radiohalogenated at a pH of 5.
  • Non-radioactive iodine onto those tyrosine residues first, before subjecting the macromolecule to radioiodination. It is highly likely that, mediated by these same endogenous deiodinases, the nonradioactive iodine on the constituent tyrosine residues would be removed, thereby restoring the original tyrosine structure and maintaining the affinity of the macromolecule for the envisioned target.
  • Non-radioactive iodination of the proteins can be simply accomplished by treating the protein with an excess of sodium iodide in the presence of an oxidizing agent such as chloramine-T.
  • VHH protein in 0.5 M sodium phosphate buffer, pH 7.4 is reacted with 15 equivalents each of sodium iodide and chloramine-T at room temperature for 5-10 min.
  • the reaction is quenched by the addition of sodium bisulphite (2 molar equivalent of chloramine-T).
  • the iodinated protein is purified by gel filtration or ultra-filtration.
  • Targeted Radiotherapy for CNS Disease An attractive strategy for treating cancers in the central nervous system (CNS) is targeted radiotherapy, which uses a vector such as a small biomolecule of the invention to selectively deliver a radionuclide to malignant cell populations.
  • An advantage of targeted radiotherapy is that one can select a radionuclide with properties that are best matched to the constraints of the intended clinical application, which for CNS tumors means selecting radiation with a tissue range that minimizes irradiation of normal CNS tissues.
  • NM neoplastic meningitis
  • Radiation dosimetry calculations indicate that radionuclides emitting short-range radiation are best for treating NM by maximizing radiation dose deposition to tumor cells while minimizing dose to spinal cord.
  • VHH molecules are also known as single -domain antibody fragments (sdAb) or nanobodies, VHH molecules are derived from Camelidae and are the smallest antigen-binding fragment of a natural antibody having a molecular weight (-15 kDa) an order of magnitude smaller than intact mAbs.
  • sdAb single -domain antibody fragments
  • VHHs are easily generated in nanomolar to picomolar affinity by cloning from immunized camels or llamas and selection by phage display panning.
  • VHHs Compared with other small protein- based targeting vectors, VHHs generally offer significant advantages in terms of thermal and chemical stability, low immunogenicity, solubility, expression yields, construction of multimers as well as the ability to recognize hidden or uncommon epitopes.
  • VHHs in both monomeric and multimeric format currently are undergoing clinical evaluation as therapeutics for a number of diseases including inflammation.
  • a panel of anti-HER2 VHHs have been labeled with a variety of radionuclides including 99m Tc, 68 Ga, 18 F, 131 I, and 177 Lu. These radiolabeled VHHs exhibited peak tumor uptake in the range of 3-6% ID/g and rapid clearance from all normal tissues except kidneys.
  • the present invention provides more potent radiolabeled biomolecules that will exhibit significantly higher tumor uptake, lower accumulation in normal tissues including the kidneys, improved radiolabeling efficiency, and are for use in targeting internalizing receptors such as HER2 and HER1.
  • Beta emitters Rationale for CNS Tumor Targeted Radiotherapy. Beta emitters such as
  • 131 I like the external beam radiation used in current CNS tumor treatments, are radiation of low energy transfer.
  • a-particles are high linear energy transfer (LET) radiation, with the result that their ability to kill cancer cells is not compromised by hypoxia, dose rate effects or cell cycle position, enhancing their attractiveness for targeted radiotherapy of CNS tumors.
  • LET linear energy transfer
  • resistance mechanisms do not limit the effectiveness of ⁇ -particles because cells have only a limited capacity to repair DNA double-strand breaks induced by ⁇ -particles, which have also been shown to kill tumor cells by apoptotic mechanisms.
  • beta and alpha emitters are encompassed by the present invention.
  • Example 8 Radiolabeled iso-SAGMB and iso-SGMIB as prosthetic agents for targeted radiotherapy of HER-2 expressing cancers
  • HER2 Human epidermal growth factor receptor 2
  • HER2 Human epidermal growth factor receptor 2
  • CNS central nervous system
  • trastuzumab the anti-HER2 monoclonal antibody trastuzumab.
  • trastuzumab frequently prolongs survival by controlling systemic disease in many patients; however, this increases the opportunity for CNS lesions, against which trastuzumab is ineffective because of poor delivery due to the blood brain barrier impermeability of this large protein.
  • HER2-positive CNS disease patients with HER2-positive CNS disease have a grim prognosis; thus, there is a dire need for treatments that can be more effective without compromising neurologic function, which can be an unfortunate side effect of nonspecific treatments including conventional radiation therapy.
  • An attractive approach for increasing the specificity of cancer treatment is targeted radiotherapy, in which a mAb or other vector is used to selectively deliver a cytotoxic radionuclide to cancer cells.
  • a-particles a radiation with a tissue range of only few cell diameters (50-80 ⁇ ), could be advantageous because it could minimize cross fire irradiation of normal tissue.
  • ⁇ -particles have a high relative biological effectiveness, requiring only a few traversals per cell to achieve its destruction.
  • trastuzumab was labeled with the 7.2-h half-life a-emitter 211 At and its cytotoxicity for 3 HER2 -expressing human breast carcinoma lines was evaluated in vitro.
  • the relative biological effectiveness of 211 At-labeled trastuzumab was about 10 times higher than that of conventional external beam therapy, with significant reduction in survival achieved with only a few 211 At atoms bound per cell.
  • a subsequent study was performed in a HER2 -positive breast carcinomatous meningitis model to evaluate the therapeutic efficacy of a single intrathecal injection of 211 At-labeled trastuzumab.
  • VHH variable domain of heavy-chain only antibodies
  • nanobodies has a molecular weight of 12-15 kDa.
  • N-succinimidyl 3-[ 2n At]astato-4-guanidinomethyl benzoate [ 211 At]SAGMB)
  • N-succinimidyl 3-[ 2n At]astato-5-guanidinomethyl benzoate wo-[ 211 At]SAGMB
  • Astatine-211 was produced on the Duke University CS-30 cyclotron via the 209 Bi(a, 2n) 211 At reaction by bombarding natural bismuth metal targets with 28 MeV a-particles. Astatine-211 was isolated from the target by dry distillation, trapped in PEEK or PTFE tubing and finally extracted with a solution of N- chlorosuccinimide (NCS) in methanol (0.2 mg/mL) as described previously.
  • NCS N- chlorosuccinimide
  • Normal -phase HPLC was performed using a 4.6 x 250 mm Partisil silica column (10 urn; Alltech, Deerfield, IL, USA), eluted in isocratic mode with a mixture of 0.2 % acetic acid in 75:25 hexanes:ethyl acetate (v/v) at a flow rate of 1 mL/min.
  • Disposable PD 10 desalting columns for gel filtration were purchased from GE Healthcare (Piscataway, NJ, USA).
  • Instant thin layer chromatography (ITLC) was performed using silica gel impregnated glass fiber sheets (Pall Corporation, East Hills, NY, USA) with PBS, pH 7.4 as the mobile phase.
  • Developed sheets were analyzed for radioactivity either using the TLC scanner described above or by cutting the sheet into small strips and counting them in an automated gamma counter. Radioactivity levels in various samples were assessed using either an LKB 1282 (Wallac, Finland) or a Perkin Elmer Wizard II (Shelton, CT, USA) automated gamma counter.
  • the anti-HER2 5F7 VHH molecule was obtained as a gift from Ablynx NV (Ghent, Belgium), was selected from phage libraries derived from llamas that had been immunized with SKBR3 human breast carcinoma cells. Its production, purification and characterization were as described previously (see Pruszynski M, Koumarianou E, Vaidyanathan G, Revets H, Devoogdt N, Lahoutte T, et al. Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody.
  • GGC glycine -glycine -cysteine
  • Cell culture reagents were purchased from Invitrogen (Grand Island, NY, USA). BT474M1 human breast carcinoma cells were grown in DMEM/F12 medium containing 10% fetal calf serum (FCS), streptomycin (100 ⁇ g/mL), and penicillin (100 IU/mL) (Sigma-Aldrich, MO, USA). Cells were cultured at 37°C in a 5% C0 2 humidified incubator.
  • FCS fetal calf serum
  • streptomycin 100 ⁇ g/mL
  • penicillin 100 IU/mL
  • the residual radioactivity was reconstituted in the HPLC mobile phase (200 ⁇ ) and injected onto a normal phase column. Procedures for isolation and deprotection were as described below for [ 211 At]SAGMB and iso- [ 211 At]SAGMB.
  • Astatine-211 in NCS/methanol (30-56 MBq) was added to a vial containing 200 ⁇ g of the required tin precursor followed by 10 ⁇ acetic acid.
  • the reaction mixture was incubated at 20°C for 30 min and methanol was evaporated with a gentle stream of argon.
  • the residual mixture was re-dissolved in 20 ⁇ of (75:25) hexanes/ethyl acetate and injected onto the normal phase HPLC column.
  • Boc protecting groups were removed by treatment with 100 ⁇ of trifluoroacetic acid (TFA) at 20°C for 10 min. To insure complete removal of TFA, the process of ethyl acetate addition (50 ⁇ ) and evaporation was performed three times. The residual radioactivity was then used as such for 5F7 VHH labeling.
  • TFA trifluoroacetic acid
  • a solution of 5F7 VHH in 0.1 M borate buffer, pH 8.5 (50 ⁇ ., 2 mg/mL) was added to the vial containing the [ 211 At]SAGMB or wo- [ 2n At] S AGMB activity and the mixture was incubated at 20°C for 20 min.
  • the labeled 5F7 VHH was purified by gel filtration over a PD-10 column eluted with phosphate buffered saline (PBS). Before use, the PD-10 column was preconditioned with human serum albumin to minimize nonspecific binding.
  • PBS phosphate buffered saline
  • Each 131 I- and 211 At-labeled 5F7 preparation was evaluated by ITLC and SDS-PAGE to determine protein associated radioactivity, and the presence of aggregates and multimeric species, respectively.
  • SDS-PAGE under non-reducing conditions and phosphor imaging were performed as previously described.
  • immunoreactive fractions of the labeled 5F7 VHH conjugates were determined by the Lindmo method using magnetic beads coated with HER2 extracellular domain, or as a negative control, bovine serum albumin (BSA). Briefly, aliquots of labeled 5F7 ( ⁇ 5 ng) were incubated with doubling concentrations of both HER2- and BSA-coated beads, and the immunoreactive fraction was calculated as the specific binding extrapolated to infinite HER2 excess.
  • BSA bovine serum albumin
  • BT474M1 breast carcinoma cells were plated in 24-well plates at a density of 8 x 10 4 cells/well and incubated at 37°C for 24 h. The cells were then allowed to acclimatize at 4°C for 30 min prior to the addition of increasing concentrations of radiolabeled 5F7 conjugates (0.1-100 nM). Cells were then incubated at 4°C for 2 h, the medium containing unbound radioactivity was removed, and the cells were washed twice with cold PBS. Finally, the cells were solubilized by treatment with IN NaOH (0.5 mL) at 37°C for 10 min. Cell-associated radioactivity was counted using an automated gamma counter.
  • mice received tail vein injections of -185 kBq each of the labeled molecules.
  • [ 211 At]SAGMB-5F7 178 MBq/mg
  • [ 131 I]SGMIB-5F7 174
  • Biodistribution was evaluated at 1 h, 2 h, 4 h, and 21 h after injection; an additional time point of 14 h was included in the second study.
  • Blood and urine were collected, and mice were killed by an overdose of isofluorane. Tumor and normal tissues were isolated, blot-dried, and weighed along with blood and urine. All tissue samples together with 5% injection standards were counted for 131 I and 211 At activity using an automated gamma counter, and the percentage of injected dose (%ID) per organ and per gram of tissue were calculated.
  • the radiochemical yield for the synthesis of [ 211 At]SAGMB-Boc 2 was similar to that reported previously when TBHP was used as the oxidant and chloroform as the solvent.
  • immunoreactive fractions were determined in paired-label format using the extracellular domain of HER2 as the molecular target.
  • the immunoreactive fractions were determined to be 81.3 ⁇ 0.9%, 83.5 ⁇ 1.1%, 81.8 ⁇ 1.4% and 84.5 ⁇ 0.8% for wo-[ 211 At]SAGMB-5F7, [ 211 At]SAGMB-5F7, wo-[ 131 I]SGMIB-5F7 and [ 131 I]SGMIB-5F7, respectively, suggesting that 5F7 VHH retained immunoreactivity to a similar degree irrespective of the prosthetic agent used.
  • the dissociation constant (K d ) values obtained from saturation binding assays performed on HER2- expressing BT474M1 human breast carcinoma cells were ⁇ 5 nM for the four labeled conjugates (FIG. 2).
  • the data of FIG. 2 was provided based on incubating cells (8 x 10 4 ) with increasing concentrations of the labeled VHH conjugates and specific cell-associated radioactivity determined as described herein. Plots were generated and Kd values calculated using GraphPad Prism software. However, significantly higher affinity binding (P ⁇ 0.05) was observed for i ' sO-[ 2n At]SAGMB-5F7 (3.0 ⁇ 0.1 nM) compared with
  • Paired-label internalization assays were performed using HER2-expressing BT474M1 cells to determine the extent of intracellular trapping of radioactivity in vitro with [ 211 At]SAGMB-5F7 and iso- [ 211 At]SAGMB-5F7 (FIG. 3), and [ 131 I]SGMIB-5F7 and wo-[ 131 I]SGMIB-5F7 (FIG. 4).
  • the data represented in FIG. 3 was generated based on two versions of the labeled 5F7, obtained from two different experiments. As shown in FIG.
  • Thyroid and stomach accumulation for both 211 At-labeled 5F7 conjugates was significantly higher than seen with their 131 I-labeled co-administered counterparts.
  • thyroid and stomach activity levels were about twofold lower for iso- [ 211 At]SAGMB-5F7 compared with [ 211 At]SAGMB-5F7, suggesting a lower degree of deastatination in vivo for /io-[ 211 At]SAGMB-5F7.
  • tumor-to-normal tissue ratios for iso- [ 211 At]SAGMB-5F7 were significantly higher than those for [ 211 At]SAGMB-5F7 in all tissues.
  • tumor-to-liver, tumor-to-blood, tumor- to-spleen and tumor-to-kidney ratios were 18 ⁇ 4, 63 ⁇ 13, 21 ⁇ 3, and 1.50 ⁇ 0.25, respectively, for iso- [ 211 At]SAGMB-5F7 at 4 h, compared with 7.31 ⁇ 1.26, 32 ⁇ 4, 7.11 ⁇ 1.47, and 0.67 ⁇ 0.08 for
  • the anti-HER2 5F7 VHH was successfully labeled with the ot-particle emitting radiohalogen 211 At using two related prosthetic agents, [ 211 At]SAGMB and /io-[ 211 At]SAGMB, designed to trap the radionuclide in HER2 -expressing cancer cells after receptor-mediated internalization through the generation of positively charged, labeled catabolites.
  • the high cytotoxicity of 211 At a-particles for HER2 expressing breast carcinoma cells has been demonstrated with 211 At-labeled trastuzumab both in vitro and in vivo in compartmental settings.
  • 211 At has many potential advantages for targeted radiotherapy, the combination of the short tissue range of its ⁇ -particles and its 7.2-h half-life necessitates the development of strategies for rapidly achieving homogeneous and prolonged delivery to cancer cells with rapid clearance from normal tissues. Most approaches for achieving this goal utilize a small molecule such as a mAb fragment; however, unlike the case with whole mAbs, 211 At -labeled mAb fragments exhibit high uptake in thyroid and stomach, indicating release of free 211 At in vivo.
  • the binding affinities for wo-[ 211 At]SAGMB-5F7 (3.0 ⁇ 0.1 nM) and [ 211 At]SAGMB-5F7 (4.5 ⁇ 0.4 nM) should be compatible with their use as targeted radiotherapeutics.
  • N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents influence of isomeric substitution on radiolabeling and target cell residualization. Nucl Med Biol 2014; 41 :802-12, which is incorporated herein by reference. Although these results suggest that the residualizing capability of «o-[*I]SGMIB is not as prolonged as that of [*I]SGMIB, this might not be a significant disadvantage with 211 At because of its 7.2-h half -life. Paired label experiments on
  • BT474M1 breast carcinoma cells permitted direct comparison of cell associated and intracellular activity for both wo-[ 2n At]SAGMB-5F7 and [ 211 At]SAGMB-5F7 to their radioiodinated counterparts.
  • ⁇ -(S-t ⁇ liodobenzoy ⁇ -Lys ⁇ -N ⁇ -maleimido-Gly ⁇ GEEEK i ⁇ -IB-Mal-D-GEEEK) was shown to be an excellent reagent for labeling intact mAb L8A4 but offered no advantages in terms of tumor uptake, and a distinct disadvantage in terms of kidney uptake, for labeling 5F7 VHH.
  • iso- [ 131 I]SGMIB-5F7 and «o-[ 211 At]SAGMB-5F7 exhibited a significant and unexpected ⁇ 1.5-fold tumor delivery advantage compared with [ 131 I]SGMIB-5F7 and [ 211 At]SAGMB-5F7 at all time points. However, this does not appear to reflect differences in residualization capacity because similar degrees of intracellular trapping were observed for both isomers in the in vitro internalization assays until the last time point.
  • stomach and thyroid radioactivity levels after injection of wo-[ 131 I]SGMIB-5F7 were lower than those for [ 131 I]SGMIB-5F7, suggesting unexpected isomer-dependent differences in the in vivo stability of these radiohalogenated sdAb conjugates.
  • Tumor-to-normal tissue ratios were generally higher for the radioiodinated conjugates compared with the astatinated versions, presumably reflecting the higher in vivo stability of the iodo versions.
  • tumor-to-normal tissue ratios were significantly higher with both radionuclides when 5F7 VHH was labeled using the iso- prosthetic agents. As summarized in Tables 1 and 2, this reflects not only some advantages in tumor uptake but also considerably lower activity levels in normal tissues, particularly with the 131 I-labeled conjugates. A possible explanation for this behavior is a mass effect wherein a certain mass of VHH molecule is needed to block nonspecific uptake of the labeled VHH in normal organs such as the liver spleen and lungs. See Xavier C, Vaneycken I, D 'Huyvetter M,Heemskerk J, Keyaerts M, Vincke C, et al.
  • VHH molecules are about 10 times smaller than intact mAbs, which may lead to more rapid degradation to species that are small enough to allow easy access to deiodinases and other enzymes such as cytochrome P450 that can lead to dehalogenation.
  • the greater metabolic stability of iSO-[ 125 I]SGMIB-5F7 compared with [ 131 I]SGMIB-5F7 could be explained by differences in the catabolism of the two conjugates and the susceptibility of the labeled catabolites towards in vivo deiodination. As summarized in a recent review, subtle differences in the design of radioiodinated compounds can lead to increased rates of deiodination.
  • a potential problem with using VHH molecules as a platform for targeted radiotherapeutics is the high accumulation and prolonged retention of radioactivity in the kidney, which could result in dose limiting renal toxicity. This behavior has been observed with radiometals such as 177 Lu as well as with some residualizing radiohalogenation agents such as 131 I-IB-Mal-D-GEEEK. For example, when 5F7-GGC was labeled using 131 I-IB-Mal-D-GEEEK, kidney levels were greater than 150% ID/g from 1-8 h after injection and about 100% ID/g at 24 h.
  • kidney radioactivity levels were high (60-100% ID/g) but decreased rapidly with renal clearance half -lives of about 1-2 h.
  • renal radioactivity levels for both the 131 I- and 211 At- labeled iso- conjugates were significantly lower than those observed for their corresponding 1,3,4-isomer conjugates at all time points with the difference in kidney retention increasing with time.
  • the renal radioactivity level observed 21 h after injection of i ' sO-[ 131 I]SGMIB-5F7 was more than 4 times lower than that for [ 131 I]SGMIB-5F7.
  • kidney radioactivity levels after injection of i ' sO-[ 2n At]SAGMB-5F7 were higher than those for co-administered i ' sO-[ 131 I]SGMIB-5F7 while renal radioactivity levels after injection of [ 211 At]SAGMB- 5F7 were lower than those for co-administered [ 131 I]SGMIB-5F7.
  • the /io-[ 211 At]SAGMB and /io-[ 131 I]SGMIB conjugates are the reagents of choice for minimizing radiation absorbed dose to the kidneys with 5F7 and potentially other VHH. If further reduction in renal radiation dose is needed, it has been shown that this can be
  • the anti-HER2 5F7 VHH can be labeled with 211 At in reasonable yields with excellent retention of affinity and immunoreactivity after labeling.
  • VHH sequences that target HER2 that are useful in the practice of the invention include those set forth in SEQ ID NOs: 1-5.
  • immunoglobin heavy chain variable region partial [Camelus dromedarius]
  • immunoglobin heavy chain variable region partial [Camelus dromedarius]
  • immunoglobin heavy chain variable region partial [Camelus dromedarius]
  • SEQ ID NO:4 immunoglobin heavy chain variable region, partial [Camelus dromedarius]
  • immunoglobin heavy chain variable region partial [Camelus dromedarius]

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Abstract

L'invention concerne des biomolécules radiomarquées et des procédés de radiomarquage de biomolécules à l'aide d'atomes d'halogène radioactif qui réduisent au minimum la perte de l'halogène radioactif résultant d'une déshalogénation in vivo, préservent l'activité biologique de la biomolécule, maximisent le maintien de la radioactivité dans les cellules cancéreuses et réduisent au minimum le maintien de la radioactivité dans les tissus normaux après une administrationin vivo. Certaines de ces biomolécules radiomarquées comprennent un atome métallique radioactif à la place, ou en plus de l'halogène radioactif. Les biomolécules présentent une affinité pour des types particuliers de cellules et peuvent se lier de façon spécifique à certaines cellules, par exemple à des cellules cancéreuses. Les biomolécules concernées comprennent des anticorps, des anticorps monoclonaux, des fragments d'anticorps, des peptides, d'autres protéines, des nanoparticules et des aptamères.
EP18718637.4A 2017-03-30 2018-03-29 Biomolécules radiomarquées et leur utilisation Pending EP3600453A1 (fr)

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CA3230848A1 (fr) * 2021-09-01 2023-03-09 Duke University Reactifs pour le marquage specifique d'un site de proteines avec des radiohalogenes, et leurs procedes de fabrication et d'utilisation
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